This application seeks to explore a new route of intracellular trafficking of growth factor- activated EGF receptor to the nucleus. Also, the capacity of a clinically relevant EGF receptor antibody to promote nuclear localization of the EGF receptor will be investigated. In brief, preliminary data show that following the addition of EGF, EGF receptors are slowly trafficked to the endoplasmic reticulum (ER). In the ER these receptors interact with the Sec61 translocon and are retrotranslocated to the cytoplasm as non-membrane bound molecules. HSP70 is required for retrotranslocation to the cytosol and likely functions by associating with transmembrane domains of the soluble EGF receptor to prevent aggregation. Knockdown of Sec61? prevents the EGF-dependent translocation of its receptor to the nucleus and the expression of cyclin D1, indicating that ER translocation is a precursor for nuclear localization and signaling function of the EGF receptor. In the first two aims, mechanistic questions are addressed regarding the cell biology and biochemistry of this pathway. In the last two aims, the pathway is explored in terms of growth control and a clinically used antibody to the EGF receptor. The first aim proposes to investigate the intracellular trafficking route by which the activated EGFR is trafficked to the ER. Experiments are proposed to determine how different cell internalization mechanisms, the Golgi, and endosomes may participate in this pathway The second aim focuses on the mechanism by which the receptor is recognized by the Sec61 translocon in the ER. This will test the possibility that the activated receptor is recognized by the known mechanism in the ER that sorts misfolded proteins for Sec-61-dependent retrotranslocation and cytosolic degradation. Also, this aim will include experiments to identify cytoplasmic and ER factors that participate in retrotranslocation, the fate(s) of translocated receptor, and the identity of EGF receptor fragments found in the ER and nucleus. This includes HSP70 and p97, an ATPase that is tyrosine phosphorylated following the addition of EGF. The third aim will determine the possible role of Sec61 in the EGF-stimulated G1?S transition. The final aim will explore the mechanism by which the clinically employed EGF receptor antibody C225 (Erbitux) is able to induce receptor trafficking to the ER and nucleus. The role of Sec61 in cell responses to C225 will be investigated also. The focus of this grant is the EGF receptor, a protein that is a rational target for several chemotherapeutic drugs that are approved for clinical use for different cancers. The application proposes investigation of this receptor in response to its normal activator, a hormone-like growth factor that stimulates cells to proliferate, as well as an antibody to the receptor that is one of the clinically approved reagents for cancer treatment.